Heterotrimeric G proteins, G12 and G13 regulate critical pathways involved in cell growth, differentiation, and apoptosis. The studies outlined here will investigate the mechanism(s) by which the alpha-subunit of G12, Galpha12 regulates distinct signaling pathways in the context of cell proliferation. The following experimental approaches will be taken: I. Analysis of the mechanism of signal coupling between Galpha12 and small GTPases in activating proliferative pathways: Studies from several laboratories including ours have shown the Galpha12- activated JNK plays a critical role in Galpha12-mediated mitogenic pathway. Galpha12 stimulates JNK-signaling modules through the small GTPases Ras, Rac, CDC42, and Rho. The mechanism(s) through which Galpha12 couples to these small GTPases in activating the kinase modules and cell proliferation will be analyzed. II. Analysis of the role of kinase signaling modules in Galpha12- stimulated cell proliferation: The preliminary studies have shown that Galpha12QL potently activates JNK and acutely inhibits p38MAPK through the differential regulation of the different MAP kinase kinases. The present study will be focused on 1) identifying molecular basis for the differential regulation of these kinases by Galpha12QL and 2) defining the role of such differential regulation in Galpha12QL-stimulated mitogenic pathway. III. Analysis of the role of small GTPases and kinases in Galpha12-stimulated COX-2 pathway: Galpha12QL stimulates the expression of several unique primary and secondary response genes including Egr-1, Jun B, and COX-2. Since the stimulation of COX- 2 can lead to the development of an autocrine loop, the mechanism through which Galpha12QL stimulates the expression of COX-2 and its role in Galpha12-mediated cell proliferation will be analyzed. The studies presented here will identify the crucial signaling pathway(s) involved in Galpha12-mediated cell proliferation and transformation. In light of the proposed causative role of Galpha12 in soft-tissue sarcoma, these studies are anticipated to provide valuable clues to the etiology of specific types of soft- tissue sarcomas such as malignant fibrous histiocytoma. The long-term goal of this project is to elucidate the mechanisms by which G proteins integrate signaling from different pathways in the regulation of cell proliferation.